Human neuronal nicotinic acetylcholine receptor α6 and β3

ABSTRACT

DNA encoding human neuronal nicotinic acetylcholine receptor alpha and beta subunits, mammalian and amphibian cells containing said DNA, methods for producing alpha and beta subunits and recombinant (i.e., isolated or substantially pure) alpha subunits (specifically α 6 ) and beta subunits (specifically β 3 ) are provided. In addition, combinations of a plurality of subunits (i.e., one or more of α 1 , α 2 , α 3 , α 4 , α 5 , α 6  and/or α 7  subunits in combination with one or more of β 3  subunits are provided.

This is a division of Ser. No. 08/484,722, filed Jun. 7, 1995, now U.S.Pat. No. 6,485,967.

This invention relates to DNA encoding human neuronal nicotinicacetylcholine receptor protein subunits, as well as the proteinsthemselves. In particular, human neuronal nicotinic acetylcholinereceptor α-subunit-encoding DNA, α-subunit proteins, β-subunit-encodingDNA, β-subunit proteins, and combinations thereof are provided.

BACKGROUND OF THE INVENTION

Ligand-gated ion channels provide a means for communication betweencells of the central nervous system. These channels convert a signal(e.g., a chemical referred to as a neurotransmitter) that is released byone cell into an electrical signal that propagates along a target cellmembrane. A variety of neurotransmitters and neurotransmitter receptorsexist in the central and peripheral nervous systems. Five families ofligand-gated receptors, including the nicotinic acetylcholine receptors(nAChRs) of neuromuscular and neuronal origins, have been identified(Stroud et al. (1990) Biochemistry 29:11009–11023). There is, however,little understanding of the manner in which the variety of receptorsgenerates different responses to neurotransmitters or to othermodulating ligands in different regions of the nervous system.

The nicotinic acetylcholine receptors (nAChRs) are multisubunit proteinsof neuromuscular and neuronal origins. These receptors form ligand-gatedion channels that mediate synaptic transmission between nerve and muscleand between neurons upon interaction with the neurotransmitteracetylcholine (ACh). Since various neuronal nicotinic acetylcholinereceptor (nAChR) subunits exist, a variety of nAChR compositions (i.e.,combinations of subunits) exist. The different nAChR compositionsexhibit different specificities for various ligands and are therebypharmacologically distinguishable. Thus, the nicotinic acetylcholinereceptors expressed at the vertebrate neuromuscular junction, invertebrate sympathetic ganglia and in the vertebrate central nervoussystem have been distinguished on the basis of the effects of variousligands that bind to different nAChR compositions. For example, theelapid α-neurotoxins that block activation of nicotinic acetylcholinereceptors at the neuromuscular junction do not block activation of someneuronal nicotinic acetylcholine receptors that are expressed on severaldifferent neuron-derived cell lines.

Muscle nAChR is a glycoprotein composed of five subunits with thestoichiometry (α)₂β(γ or ε)δ. Each of the subunits has a mass of about50–60 kilodaltons (kd) and is encoded by a different gene. The (α)₂β(γor ε)δ complex forms functional receptors containing two ligand bindingsites and a ligand-gated transmembrane channel. Upon interaction with acholinergic agonist, muscle nicotinic nAChRs conduct sodium ions. Theinflux of sodium ions rapidly short-circuits the normal ionic gradientmaintained across the plasma membrane, thereby depolarizing themembrane. By reducing the potential difference across the membrane, achemical signal is transduced into an electrical signal at theneuromuscular junction that induces muscle contraction.

Functional muscle nicotinic acetylcholine receptors have been formedwith αβδγ subunits, αβγ subunits, αβδ subunits, αδγ subunits or αδsubunits, but not with only one subunit (see e.g., Kurosaki et al.(1987) FEBS Lett. 214: 253–258; Camacho et al. (1993) J. Neuroscience13:605–613). In contrast, functional neuronal nAChRs can be formed fromu subunits alone or combinations of α and β subunits. The larger αsubunit is generally believed to be a ACh-binding subunit and the lowermolecular weight β subunit is generally believed to be the structuralsubunit, although it has not been definitively demonstrated that the βsubunit does not have the ability to bind ACh or participate in theformation of the ACh binding site. Each of the subunits whichparticipate in the formation of a functional ion channel are, to theextent they contribute to the structure of the resulting channel,“structural” subunits, regardless of their ability (or inability) tobind ACh. Neuronal nAChRs, which are also ligand-gated ion channels, areexpressed in ganglia of the autonomic nervous system and in the centralnervous system (where they mediate signal transmission), inpost-synaptic locations (where they modulate transmission), and in pre-and extra-synaptic locations (where they modulate neurotransmission andmay have additional functions; Wonnacott et al., In: Progress in BrainResearch (A. Nordberg et al., (Eds) Elsevier, Amsterdam) 157–163(1990)).

DNA encoding nAChRs has been isolated from several sources. Based on theinformation available from such work, it has been evident for some timethat nAChRs expressed in muscle, in autonomic ganglia, and in thecentral nervous system are functionally diverse. This functionaldiversity could be due, at least in part, to the large number ofdifferent nAChR subunits which exist. There is an incompleteunderstanding, however, of how (and which) nAChR subunits combine togenerate unique nAChR subtypes, particularly in neuronal-cells. Indeed,there is evidence that only certain nAChR subtypes may be involved indiseases such as Alzheimer's disease. Moreover, it is not clear whethernAChRs from analogous tissues or cell types are similar across species.

Accordingly, there is a need for the isolation and characterization ofDNAs encoding each human neuronal nAChR subunit, recombinant cellscontaining such subunits and receptors prepared therefrom. In order tostudy the function of human neuronal nAChRs and to obtaindisease-specific pharmacologically active agents, there is also a needto obtain isolated (preferably purified) human neuronal nAChRs, andisolated (preferably purified) human neuronal nAChR subunits. Inaddition, there is also a need to develop assays to identify suchpharmacologically active agents.

The availability of such DNAs, cells, receptor subunits and receptorcompositions will eliminate the uncertainty of speculating as to humanneuronal nAChR structure and function based on predictions drawn fromnon-human nAChR data, or human or non-human muscle or ganglia nAChRdata.

Therefore, it is an object herein to isolate and characterize DNAencoding subunits of human neuronal nicotinic acetylcholine receptors.It is also an object herein to provide methods for recombinantproduction of human neuronal nicotinic acetylcholine receptor subunits.It is also an object herein to provide purified receptor subunits and toprovide methods for screening compounds to identify compounds thatmodulate the activity of human neuronal nAChRs.

These and other objects will become apparent to those of skill in theart upon further study of the specification and claims.

BRIEF DESCRIPTION OF THE INVENTION

In accordance with the present invention, there are provided isolatedDNAs encoding novel human alpha and beta subunits of neuronal nAChRs. Inparticular, isolated DNA encoding human α₆ and β₃ subunits of neuronalnAChRs are provided. Messenger RNA and polypeptides encoded by theabove-described DNA are also provided.

Further in accordance with the present invention, there are providedrecombinant human neuronal nicotinic nAChR subunits, including α₆ and β₃subunits, as well as methods for the production thereof. In addition,recombinant human neuronal nicotinic acetylcholine receptors containingat least one human neuronal nicotinic nAChR subunit are also provided,as well as methods for the production thereof. Further provided arerecombinant neuronal nicotinic nAChRs that contain a mixture of one ormore nAChR subunits encoded by a host cell, and one or more nAChRsubunits encoded by heterologous DNA or RNA (i.e., DNA or RNA asdescribed -herein that has been introduced into the host cell), as wellas methods for the production thereof.

Plasmids containing DNA encoding the above-described subunits are alsoprovided. Recombinant cells containing the above-described DNA, mRNA orplasmids are also provided herein. Such cells are useful, for example,for replicating DNA, for producing human nAChR subunits and recombinantreceptors, and for producing cells that express receptors containing oneor more human subunits.

The DNA, mRNA, vectors, receptor subunits, receptor subunit combinationsand cells provided herein permit production of selected neuronalnicotinic nAChR receptor subtypes and specific combinations thereof, aswell as antibodies to said receptor subunits. This provides a means toprepare synthetic or recombinant receptors and receptor subunits thatare substantially free of contamination from many other receptorproteins whose presence can interfere with analysis of a single nAChRsubtype. The availability of desired receptor subtypes makes it possibleto observe the effect of a drug substance on a particular receptorsubtype and to thereby perform initial in vitro screening of the drugsubstance in a test system that is specific for humans and specific fora human neuronal nicotinic nAChR subtype.

The ability to screen drug substances in vitro to determine the effectof the drug on specific receptor compositions should permit thedevelopment and screening of receptor subtype-specific ordisease-specific drugs. Also, testing of single receptor subunits orspecific receptor subtype combinations with a variety of potentialagonists or antagonists provides additional information with respect tothe function and activity of the individual subunits and should lead tothe identification and design of compounds that are capable ofvery-specific interaction with one or more of the receptor subunits orreceptor subtypes. The resulting drugs should exhibit fewer unwantedside effects than drugs identified by screening with cells that expressa variety of subtypes.

Further in relation to drug development and therapeutic treatment ofvarious disease states, the availability of DNAs encoding human neuronalnAChR subunits enables identification of any alterations in such genes(e.g., mutations) which may correlate with the occurrence of certaindisease states. In addition, the creation of animal models of suchdisease states becomes possible, by specifically introducing suchmutations into synthetic DNA sequences which can then be introduced intolaboratory animals or in vitro assay systems to determine the effectsthereof.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 presents a restriction map of a cytomegalovirus (CMV)promoter-based vector, pcDNA3-KEalpha6.3, which contains an α₆-encodingsequence as an EcoRI insert.

FIG. 2 presents a restriction map of a CMV promoter-based vector,pcDNA3-KEbeta3.2, which contains a β₃-encoding sequence as an EcoRIinsert.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention, we have isolated andcharacterized DNAs encoding novel human alpha and beta subunits ofneuronal nAChRs. Specifically, isolated DNAs encoding human α₆ and β3subunits of neuronal nAChRs are described herein. Recombinant messengerRNA (mRNA) and recombinant polypeptides encoded by the above-describedDNA are also provided.

As used herein, isolated (or substantially pure) as a modifier of DNA,RNA, polypeptides or proteins means that the DNA, RNA, polypeptides orproteins so designated have been separated from their in vivo cellularenvironments through the efforts of human beings. Thus as used herein,isolated (or substantially pure) DNA refers to DNAs purified accordingto standard techniques employed by those skilled in the art (see, e.g.,Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed.,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).

Similarly, as used herein, “recombinant” as a modifier of DNA, RNA,polypeptides or proteins means that the DNA, RNA, polypeptides orproteins so designated have been prepared by the efforts of humanbeings, e.g., by cloning, recombinant expression, and the like. Thus asused herein, recombinant proteins, for example, refers to proteinsproduced by a recombinant host, expressing DNAs which have been added tothat host through the efforts of human beings.

As used herein, a human alpha subunit gene is a gene that encodes analpha subunit of a human neuronal nicotinic acetylcholine receptor. Thealpha subunit is a subunit of the nAChR to which ACh binds. Assignmentof the name “alpha” to a putative nAChR subunit, according to Deneris etal. [Tips (1991) 12:34–40] is based on the conservation of adjacentcysteine residues in the presumed extracellular domain of the subunitthat are the homologues of cysteines 192 and 193 of the Torpedo alphasubunit (see Noda et al. (1982) Nature 299:793–797). As used herein, analpha subunit subtype refers to a human neuronal nAChR subunit that isencoded by DNA that hybridizes under high stringency conditions to atleast one of the neuronal nAChR alpha subunit-encoding DNAs (ordeposited clones) disclosed herein. An alpha subunit generally binds toACh under physiological conditions and at physiological concentrationsand, in the optional presence of a beta subunit (i.e., some alphasubunits are functional alone, while others require the presence of abeta subunit), generally forms a functional nAChR as assessed by methodsdescribed herein or known to those of skill in this art.

Also contemplated are alpha subunits encoded by DNAs that encode alphasubunits as defined above, but that by virtue of degeneracy of thegenetic code do not necessarily hybridize to the disclosed DNA ordeposited clones under specified hybridization conditions. Such subunitsalso form a functional receptor, as assessed by the methods describedherein or known to those of skill in the art, generally with one or morebeta subunit subtypes. Typically, unless an alpha subunit is encoded byRNA that arises from alternative splicing (i.e., a splice variant),alpha-encoding DNA and the alpha subunit encoded thereby sharesubstantial sequence homology with at least one of the alpha subunitDNAs (and proteins encoded thereby) described or deposited herein. It isunderstood that DNA or RNA encoding a splice variant may overall shareless than 90% homology with the DNA or RNA provided herein, but includeregions of nearly 100% homology to a DNA fragment or deposited clonedescribed herein, and encode an open reading frame that includes startand stop codons and encodes a functional alpha subunit.

As used herein, a splice variant refers to variant nAChRsubunit-encoding nucleic acid(s) produced by differential processing ofprimary transcript(s) of genomic DNA, resulting in the production ofmore than one type of mRNA. cDNA derived from differentially processedgenomic DNA will encode nAChR subunits that have regions of completeamino acid identity and regions having different amino acid sequences.Thus, the same genomic sequence can lead to the production of multiple,related mRNAs and proteins. Both the resulting mRNAs and proteins arereferred to herein as “splice variants”.

Stringency of hybridization is used herein to refer to conditions underwhich polynucleic acid hybrids are stable. As known to those of skill inthe art, the stability of hybrids is reflected in the meltingtemperature (T_(m)) of the hybrids. T_(m) can be approximated by theformula:81.5° C.−16.6(log₁₀[Na⁺])+0.41(% G+C)−600/l,where l is the length of the hybrids in nucleotides. T_(m) decreasesapproximately 1–1.5° C. with every 1% decrease in sequence homology. Ingeneral, the stability of a hybrid is a function of sodium ionconcentration and temperature. Typically, the hybridization reaction isperformed under conditions of lower stringency, followed by washes ofvarying, but higher, stringency. Reference to hybridization stringencyrelates to such washing conditions. Thus, as used herein:

-   -   (1) HIGH STRINGENCY conditions, with respect to fragment        hybridization, refer to conditions that permit hybridization of        only those nucleic acid sequences that form stable hybrids in        0.018M NaCl at 65° C. (i.e., if a hybrid is not stable in 0.018M        NaCl at 65° C., it will not be stable under high stringency        conditions, as contemplated herein). High stringency conditions        can be provided, for example, by hybridization in 50% formamide,        5× Denhardt's solution, 5×SSPE, 0.2% SDS, 200 μg/ml denatured        sonicated herring sperm DNA, at 42° C., followed by washing in        0.1×SSPE, and 0.1% SDS at 65° C.;    -   (2) MODERATE STRINGENCY conditions, with respect to fragment        hybridization, refer to conditions equivalent to hybridization        in 50% formamide, 5× Denhardt's solution, 5×SSPE, 0.2% SDS, 200        μg/ml denatured sonicated herring sperm DNA, at 42° C., followed        by washing in 0.2×SSPE, 0.2% SDS, at 60° C.;    -   (3) LOW STRINGENCY conditions, with respect to fragment        hybridization, refer to conditions equivalent to hybridization        in 10% formamide, 5× Denhardt's solution, 6×SSPE, 0.2% SDS, 200        μg/ml denatured sonicated herring sperm DNA, followed by washing        in 1× SSPE, 0.2% SDS, at 50° C.; and    -   (4) HIGH STRINGENCY conditions, with respect to oligonucleotide        (i.e., synthetic DNA≦about 30 nucleotides in length)        hybridization, refer to conditions equivalent to hybridization        in 10% formamide, 5× Denhardt's solution, 6×SSPE, 0.2% SDS, 200        μg/ml denatured sonicated herring sperm DNA, at 42° C., followed        by washing in 1×SSPE, and 0.2% SDS at 50° C.        It is understood that these conditions may be duplicated using a        variety of buffers and temperatures and that they are not        necessarily precise.

Denhardt's solution and SSPE (see, e.g., Sambrook et al., supra) arewell known to those of skill in the art as are other suitablehybridization buffers. For example, SSPE is pH 7.4 phosphate-buffered0.18M NaCl. SSPE can be prepared, for example, as a 20× stock solutionby dissolving 175.3 g of NaCl, 27.6 g of NaH₂PO₄ and 7.4 g EDTA in 800ml of water, adjusting the pH to 7.4, and then adding water to 1 liter.Denhardt's solution (see, Denhardt (1966) Biochem. Biophys. Res. Commun.23:641) can be prepared, for example, as a 50× stock solution by mixing5 g Ficoll (Type 400, Pharmacia LKB Biotechnology, INC., PiscatawayN.J.), 5 g of polyvinylpyrrolidone, 5 g bovine serum albumin (FractionV; Sigma, St. Louis Mo.) water to 500 ml and filtering to removeparticulate matter.

As used herein, the phrase “substantial sequence homology” refers tonucleotide sequences which share at least about 90% identity, and aminoacid sequences which typically share more than 95% amino acid identity.It is recognized, however, that proteins (and DNA or mRNA encoding suchproteins) containing less than the above-described level of homologyarising as splice variants or that are modified by conservative aminoacid substitutions (or substitution of degenerate codons) arecontemplated to be within the scope of the present invention.

The phrase “substantially the same” is used herein in reference to thenucleotide sequence of DNA, the ribonucleotide sequence of RNA, or theamino acid sequence of protein, that have slight and non-consequentialsequence variations from the actual sequences disclosed herein. Speciesthat are substantially the same are considered to be equivalent to thedisclosed sequences and as such are within the scope of the appendedclaims. In this regard, “slight and non-consequential sequencevariations” mean that sequences that are substantially the same as theDNA, RNA, or proteins disclosed and claimed herein are functionallyequivalent to the human-derived sequences disclosed and claimed herein.Functionally equivalent sequences will function in substantially thesame manner to produce substantially the same compositions as thehuman-derived nucleic acid and amino acid compositions disclosed andclaimed herein. In particular, functionally equivalent DNAs encodehuman-derived proteins that are the same as those disclosed herein orthat have conservative amino acid variations, such as substitution of anon-polar residue for another non-polar residue or a charged residue fora similarly charged residue. These changes include those recognized bythose of skill in the art as those that do not substantially alter thetertiary structure of the protein.

As used herein, “α₆ subunit DNA” refers to DNA encoding a neuronalnicotinic acetylcholine receptor subunit of the same name. Such DNA canbe characterized in a number of ways, for example

said DNA may encode the amino acid sequence set forth in SEQ ID NO: 2,or

said DNA may encode the amino acid sequence encoded by clonepcDNA3-KEalpha6.3 (as illustrated in FIG. 1).

Presently preferred α₆-encoding DNAs can be characterized as follows

said DNA may hybridize to the coding sequence set forth in SEQ ID NO: 1(preferably to substantially the entire coding sequence thereof, i.e.,nucleotides 143–1624) under high stringency conditions, or

said DNA may hybridize under high stringency conditions to the sequence(preferably to substantially the entire sequence) of the α₆-encodinginsert of clone pcDNA3-KEalpha6.3 (as illustrated in FIG. 1).

Especially preferred α₆-encoding DNAs of the invention are characterizedas follows

DNA having substantially the same nucleotide sequence as the α₆ codingregion set forth in SEQ ID NO:1 (i.e., nucleotides 143–1624 thereof), or

DNA having substantially the same nucleotide sequence as the α₆-encodinginsert of clone pcDNA3-KEalpha6.3 (as illustrated in FIG. 1).

Typically, unless an α₆ subunit arises as a splice variant, α₆-encodingDNA will share substantial sequence homology (i.e., greater than about90%), with the α₆ DNAs described herein. DNA or RNA encoding a splicevariant may share less than 90% overall sequence homology with the DNAor RNA provided herein, but such a splice variant would include regionsof nearly 100% homology to the above-described DNAs.

As used herein, a human beta subunit gene is a gene that encodes a betasubunit of a human neuronal nicotinic acetylcholine receptor. Assignmentof the name “beta” to a putative neuronal nAChR subunit, according toDeneris et al. supra, is based on the lack of adjacent cysteine residues(which are characteristic of alpha subunits). The beta subunit isfrequently referred to as the structural nAChR subunit (although it ispossible that beta subunits also have ACh binding properties).Combination of the appropriate beta subunit(s) with appropriate alphasubunit(s) leads to the formation of a functional receptor. As usedherein, a beta subunit subtype refers to a neuronal nAChR subunit thatis encoded by DNA that hybridizes under high stringency conditions to atleast one of the neuronal nAChR-encoding DNAs (or deposited clones)disclosed herein. A beta subunit may form a functional nAChR, asassessed by methods described herein or known to those of skill in thisart, with appropriate alpha subunit subtype(s).

Also contemplated are beta subunits encoded by DNAs that encode betasubunits as defined above, but that by virtue of degeneracy of thegenetic code do not necessarily hybridize to the disclosed DNA ordeposited clones under the specified hybridization conditions. Suchsubunits may also form functional receptors, as assessed by the methodsdescribed herein or known to those of skill in the art, in combinationwith appropriate alpha subunit subtype(s). Typically, unless a betasubunit is encoded by RNA that arises as a splice variant, beta-encodingDNA and the beta subunit encoded thereby share substantial sequencehomology with the beta-encoding DNA and beta subunit protein describedherein. It is understood that DNA or RNA encoding a splice variant mayshare less than 90% overall homology with the DNA or RNA providedherein, but such DNA will include regions of nearly 100% homology to theDNA described herein.

As used herein, “β₃ subunit DNA” refers to DNA encoding a neuronalnicotinic acetylcholine receptor subunit of the same name. Such DNA canbe characterized in a number of ways, for example, the nucleotides ofsaid DNA may encode the amino acid sequence set forth in SEQ ID NO:4.Presently preferred β₃-encoding DNAs can be characterized as DNA whichhybridizes under high stringency conditions to the coding sequence setforth in SEQ ID NO:3 (preferably to substantially the entire codingsequence thereof, i.e., nucleotides 98–1472). Especially preferredβ₃-encoding DNAs of the invention are characterized as havingsubstantially the same nucleotide sequence as set forth in SEQ ID NO:3.

Typically, unless a β₃ subunit arises as a splice variant, β₃-encodingDNA will share substantial sequence homology (greater than about 90%)with the β₃ DNAs described herein. DNA or RNA encoding a splice variantmay share less than 90% overall sequence homology with the DNA or RNAprovided herein, but such DNA would include regions of nearly 100%homology to the above-described DNA.

DNA encoding human neuronal nicotinic nAChR alpha and beta subunits maybe isolated by screening suitable human cDNA or human genomic librariesunder suitable hybridization conditions with DNA disclosed herein(including nucleotides derived from SEQ ID NOs:1 or 3). Suitablelibraries can be prepared from neuronal tissue samples, basal ganglia,thalamus, hypothalamus, and the like. The library is preferably screenedwith a portion of DNA including the entire subunit-encoding sequencethereof, or the library may be screened with a suitable probe.

As used herein with reference to human α₆ subunits, a probe issingle-stranded DNA or RNA that has a sequence of nucleotides thatincludes at least 27 contiguous bases that are the same as (or thecomplement of) any 27 bases set forth in SEQ ID NO:1. As used hereinwith reference to human β₃ subunits, a probe is single-stranded DNA orRNA that has a sequence of nucleotides that includes at least 28contiguous bases that are the same as (or the complement of) any 28bases derived from the first 105 nucleotides of signal sequence/codingsequence set forth in SEQ ID NO:3. Preferred regions from which toconstruct probes include 5′ and/or 3′ coding sequences, sequencespredicted to encode transmembrane domains, sequences predicted to encodethe cytoplasmic loop, signal sequences, acetylcholine (Ach) andα-bungarotoxin (α-bgtx) binding sites, and the like. Amino acids thatcorrespond to residues 190–198 of the Torpedo nAChR α subunit (seeKarlin (1993) Curr. Opin. Neurobiol. 3, 299–309) are typically involvedin ACh and α-bgtx binding. The approximate amino acid residues whichcomprise such regions for other preferred probes are set forth in thefollowing table:

Subunit Signal Seqence TMD1* TMD2 TMD3 TMD4 Cytoplasmic Loop α₆ 1–30240–265 272–294 301–326 458–483 327–457 β₃ 1–20 231–258 265–287 293–318421–446 319–420 *TMD = transmembrane domainAlternatively, portions of the DNA can be used as primers to amplifyselected fragments in a particular library.

After screening the library, positive clones are identified by detectinga hybridization signal; the identified clones are characterized byrestriction enzyme mapping and/or DNA sequence analysis, and thenexamined, by comparison with the sequences set forth herein or with thedeposited clones described herein, to ascertain whether they include DNAencoding a complete alpha or beta subunit. If the selected clones areincomplete, they may be used to rescreen the same or a different libraryto obtain overlapping clones. If desired, the library can be rescreenedwith positive clones until overlapping clones that encode an entirealpha or beta subunit are obtained. If the library is a cDNA library,then the overlapping clones will include an open reading frame. If thelibrary is genomic, then the overlapping clones may include exons andintrons. In both instances, complete clones may be identified bycomparison with the DNA and encoded proteins provided herein.

Complementary DNA clones encoding various subtypes of human neuronalnAChR alpha and beta subunits have been isolated. Each subtype of thesubunit appears to be encoded by a different gene. The DNA clonesprovided herein may be used to isolate genomic clones encoding eachsubtype and to isolate any splice variants by screening librariesprepared from different neural tissues. Nucleic acid amplificationtechniques, which are well known in the art, can be used to locatesplice variants of human neuronal nAChR subunits. This is accomplishedby employing oligonucleotides based on DNA sequences surroundingdivergent sequence(s) as primers for amplifying human RNA or genomicDNA. Size and sequence determinations of the amplification products canreveal the existence of splice variants. Furthermore, isolation of humangenomic DNA sequences by hybridization can yield DNA containing multipleexons, separated by introns, that correspond to different splicevariants of transcripts encoding human neuronal nAChR subunits.

It has been found that not all subunit subtypes are expressed in allneural tissues or in all portions of the brain. Thus, in order toisolate cDNA encoding particular subunit subtypes or splice variants ofsuch subtypes, it is preferable to screen libraries prepared fromdifferent neuronal or neural tissues. Preferred libraries for obtainingDNA encoding each subunit include: substantia nigra, thalamus orhypothalamus to isolate human α₆-encoding DNA and substantia nigra orthalamus to isolate human β₃-encoding DNA.

The above-described nucleotide sequences can be incorporated intovectors for further manipulation. As used herein, vector (or plasmid)refers to discrete elements that are used to introduce heterologous DNAinto cells for either expression or replication thereof. Selection anduse of such vehicles are well within the level of skill of the art.

An expression vector includes vectors capable of expressing DNAs thatare operatively linked with regulatory sequences, such as promoterregions, that are capable of effecting expression of such DNA fragments.Thus, an expression vector refers to a recombinant DNA or RNA construct,such as a plasmid, a phage, recombinant virus or other vector that, uponintroduction into an appropriate host cell, allows expression of DNAcloned into the appropriate site on the vector. Appropriate expressionvectors are well known to those of skill in the art and include thosethat are replicable in eukaryotic cells and/or prokaryotic cells andthose that remain episomal or those which integrate into the host cellgenome. Presently referred plasmids for expression of invention nAChRsubunits in eukaryotic host cells, particularly mammalian cells, includecytomegalovirus (CMV), Simian virus 40 (SV40) and mouse mammary tumorvirus (MMTV) promoter-containing vectors such as pCMV, pcDNA1, pcDNA3,pZeoSV, PCEP4, pMAMneo, pMAMhyg, and the like.

As used herein, a promoter region refers to a segment of DNA thatcontrols transcription of DNA to which it is operatively linked. Thepromoter region includes specific sequences that are sufficient for RNApolymerase recognition, binding and transcription initiation. Thisportion of the promoter region is referred to as the promoter. Inaddition, the promoter region includes sequences that modulate thisrecognition, binding and transcription initiation activity of RNApolymerase. These sequences may be cis acting or may be responsive totrans acting factors. Promoters, depending upon the nature of theregulation, may be constitutive or regulated. Exemplary promoterscontemplated for use in the practice of the present invention includethe SV40 early promoter, the cytomegalovirus (CMV) promoter, the mousemammary tumor virus (MMTV) steroid-inducible promoter, Moloney murineleukemia virus (MMLV) promoter, and the like.

As used herein, the term “operatively linked” refers to the functionalrelationship of DNA with regulatory and effector sequences ofnucleotides, such as promoters, enhancers, transcriptional andtranslational stop sites, and other signal sequences. For example,operative linkage of DNA to a promoter refers to the physical andfunctional relationship between the DNA and the promoter such that thetranscription of such DNA is initiated from the promoter by an RNApolymerase that specifically recognizes, binds to and transcribes theDNA. In order to optimize expression and/or in vitro transcription, itmay be necessary to remove or alter 5′ untranslated portions of theclones to remove extra, potential alternative translation initiation(i.e., start) codons or other sequences that interfere with or reduceexpression, either at the level of transcription or translation.Alternatively, consensus ribosome binding sites (see, for example, Kozak(1991) J. Biol. Chem. 266:19867–19870) can be inserted immediately 5′ ofthe start codon to enhance expression. The desirability of (or need for)such modification may be empirically determined.

As used herein, expression refers to the process by which polynucleicacids are transcribed into mRNA and translated into peptides,polypeptides, or proteins. If the polynucleic acid is derived fromgenomic DNA, expression may, if an appropriate eukaryotic host cell ororganism is selected, include splicing of the mRNA.

Particularly preferred vectors for transfection of mammalian cells arethe SV40 promoter-based expression vectors, such as pZeoSV (Invitrogen,San Diego, Calif.) CMV promoter-based vectors such as pcDNA1, pcDNA3,pCEP4 (Invitrogen, San Diego, Calif.), and MMTV promoter-based vectorssuch as pMAMneo (Clontech, Inc.).

Full-length DNAs encoding human neuronal nAChR subunits have beeninserted into vector pcDNA3, a pUC19-based mammalian cell expressionvector containing the CMV promoter/enhancer, a polylinker downstream ofthe CMV promoter/enhancer, followed by a bovine growth hormone (BGH)polyadenylation signal. Placement of nAChR subunit DNA between the CMVpromoter and BGH polyadenylation signal provides for constitutiveexpression of the foreign DNA in a mammalian host cell transfected withthe construct. For inducible expression of human nAChR subunit-encodingDNA in a mammalian cell, the DNA can be inserted into a plasmid such aspMAMneo. This plasmid contains the mouse mammary tumor virus (MMTV)promoter for steroid-inducible expression of operatively associatedforeign DNA. If the host cell does not express endogenous glucocorticoidreceptors required for uptake of glucorcorticoids (i.e., inducers of theMMTV promoter) into the cell, it is necessary to additionally transfectthe cell with DNA encoding the glucocorticoid receptor (ATCC accessionno. 67200).

In accordance with another embodiment of the present invention, thereare provided cells containing the above-described polynucleic acids(i.e., DNA or mRNA). Such host cells as bacterial, yeast and mammaliancells can be used for replicating DNA and producing nAChR subunit(s).Methods for constructing expression vectors, preparing in vitrotranscripts, transfecting DNA into mammalian cells, injecting oocytes,and performing electrophysiological and other analyses for assessingreceptor expression and function as described herein are also describedin PCT Application Nos. PCT/US91/02311, PCT/US91/05625 andPCT/US92/11090, and in co-pending U.S. application Ser. Nos. 07/504,455,07/563,751 and 07/812,254. The subject matter of these applications arehereby incorporated by reference herein in their entirety.

Incorporation of cloned DNA into a suitable expression vector,transfection of eukaryotic cells with one or a combination of expressionconstructs encoding one or more distinct genes or with linear DNA, andselection of transfected cells are well known in the art (see, e.g.,Sambrook et al. (1989) supra). Heterologous DNA may be introduced intohost cells by any method known to those of skill in the art, such astransfection with an expression construct encoding the heterologous DNAby CaPO₄ precipitation (see, e.g., Wigler et al. (1979) Proc. Natl.Acad. Sci. 76:1373–1376). Recombinant cells can then be cultured underconditions whereby the subunit(s) encoded by the DNA is (are) expressed.Preferred cells include mammalian cells (e.g., HEK 293, CHO and Ltk⁻cells), yeast cells (e.g., methylotrophic yeast cells, such as Pichiapastoris), bacterial cells (e.g., Escherichia coli), and the like.

While the DNA provided herein may be expressed in any eukaryotic cell,including yeast cells (such as, for example, P. pastoris (see U.S. Pat.Nos. 4,882,279, 4,837,148, 4,929,555 and 4,855,231), Saccharomycescerevisiae, Candida tropicalis, Hansenula polymorpha, and the like),mammalian expression systems, including commercially available systemsand other such systems known to those of skill in the art, forexpression of DNA encoding the human neuronal nicotinic nAChR subunitsprovided herein are presently preferred. Xenopus oocytes are preferredfor expression of RNA transcripts of the DNA.

In preferred embodiments, DNA is ligated into a vector, and theresulting construct is introduced into suitable host cells to producetransformed cell lines that express a specific human neuronal nAChRreceptor subtype, or specific combinations of subtypes. The resultingcell lines can then be produced in quantity for reproduciblequantitative analysis of the effects of drugs on receptor function. Inother embodiments, mRNA may be produced by in vitro transcription of DNAencoding each subunit. This mRNA, either from a single subunit clone orfrom a combination of clones, can then be injected into Xenopus oocyteswhere the mRNA directs the synthesis of the human receptor subunits,which then form functional receptors. Alternatively, thesubunit-encoding DNA can be directly injected into oocytes forexpression of functional receptors. The transfected mammalian cells orinjected oocytes may then be used in the methods of drug screeningprovided herein.

Cloned full-length DNA encoding any of the subunits of human neuronalnicotinic nAChR may be introduced into a plasmid vector for expressionin a eukaryotic cell. Such DNA may be genomic DNA or cDNA. Host cellsmay be transfected with one or a combination of plasmids, each of whichencodes at least one human neuronal nAChR subunit.

Eukaryotic cells in which DNA or RNA may be introduced include any cellsthat are transfectable by such DNA or RNA or into which such DNA or RNAmay be injected. Preferred cells are those that can be transiently orstably transfected and also express the DNA and RNA. Presently mostpreferred cells are those that can form recombinant or heterologoushuman neuronal nicotinic nAChRs comprising one or more subunits encodedby the heterologous DNA. Such cells may be identified empirically orselected from among those known to be readily transfected or injected.

Exemplary cells for introducing DNA include cells of mammalian origin(e.g., COS cells, mouse L cells, Chinese hamster ovary (CHO) cells,human embryonic kidney (HEK) cells, GH3 cells and other such cells knownto those of skill in the art), amphibian cells (e.g., Xenopus laevisoöcytes), yeast cells (e.g., Saccharomyces cerevisiae, Pichia pastoris),and the like. Exemplary cells for expressing injected RNA transcriptsinclude Xenopus laevis oöcytes. Cells that are preferred fortransfection of DNA are known to those of skill in the art or may beempirically identified, and include HEK 293 (which are available fromATCC under accession #CRL 1573); Ltk⁻ cells (which are available fromATCC under accession #CCL1.3); COS-7 cells (which are available fromATCC under accession #CRL 1651); and GH₃ cells (which are available fromATCC under accession #CCL82.1). Presently preferred cells include GH3cells and HEK 293 cells, particularly HEK 293 cells that have beenadapted for growth in suspension and that can be frozen in liquidnitrogen and then thawed and regrown. HEK 293 cells are described, forexample, in U.S. Pat. No. 5,024,939 to Gorman (see, also, Stillman etal. (1985) Mol. Cell. Biol. 5:2051–2060).

DNA may be stably incorporated into cells or may be transientlyintroduced using methods known in the art. Stably transfected mammaliancells may be prepared by transfecting cells either with one or moreexpression constructs carrying DNA encoding nAChR subunits and aseparate expression vector carrying a selectable marker gene (e.g., thegene for neomycin resistance, zeocin resistance, hygromycin resistanceand the like) or with one or more expression constructs which carry boththe DNA encoding nAChR subunit and the selectable marker, and growingthe transfected cells under conditions selective for cells expressingthe marker gene(s). To produce such cells, the cells should betransfected with a sufficient concentration of subunit-encoding nucleicacids to form human neuronal nAChRs that contain the human subunitsencoded by heterologous DNA. The precise amounts and ratios of DNAencoding the subunits may be empirically determined and optimized for aparticular combination of subunits, cells and assay conditions.Recombinant cells that express neuronal nAChR containing subunitsencoded only by the heterologous DNA or RNA are especially preferred.

Heterologous DNA may be maintained in the cell as an episomal element ormay be integrated into chromosomal DNA of the cell. The resultingrecombinant cells may then be cultured or subcultured (or passaged, inthe case of mammalian cells) from such a culture or a subculturethereof. Methods for transfection, injection and culturing recombinantcells are known to the skilled artisan. Similarly, the human neuronalnicotinic nAChR subunits may be purified using protein purificationmethods known to those of skill in the art. For example, antibodies orother ligands that specifically bind to one or more of the subunits maybe used for affinity purification of the subunit or human neuronalnAChRs containing the subunits.

In accordance with one embodiment of the present invention, methods forproducing cells that express human neuronal nAChR subunits andfunctional receptors are also provided. In one such method, host cellsare transfected with DNA encoding at least one alpha subunit of aneuronal nAChR and at least one beta subunit of a neuronal nAChR. Usingmethods such as northern blot or slot blot analysis, transfected cellsthat contain alpha and/or beta subunit encoding DNA or RNA can be,selected. Transfected cells are also analyzed to identify those thatexpress nAChR protein. Analysis can be carried out, for example, bymeasuring the ability of cells to bind acetylcholine, nicotine, or anAChR agonist, compared to the nicotine binding ability of untransfectedhost cells or other suitable control cells, by electrophysiologicallymonitoring the currents through the cell membrane in response to a nAChRagonist, and the like.

In particularly preferred aspects, eukaryotic cells which containheterologous DNAs express such DNA and form recombinant functionalneuronal nAChR(s). In more preferred aspects, recombinant neuronal nAChRactivity is readily detectable because it is a type that is absent fromthe untransfected host cell or is of a magnitude not exhibited in theuntransfected cell. Such cells that contain recombinant receptors couldbe prepared, for example, by causing cells transformed with DNA encodingthe human neuronal nicotinic nAChR α₆ and β₃ subunits to express thecorresponding proteins in the presence or absence of one or more alphaand/or beta nAChR subunits. The resulting synthetic or recombinantreceptor would contain the α₆ and β₃ nAChR subunits. Such a receptorwould be useful for a variety of applications, e.g., as part of an assaysystem free of the interferences frequently present in prior art assaysystems employing non-human receptors or human tissue preparations.Furthermore, testing of single receptor subunits with a variety ofpotential agonists or antagonists would provide additional informationwith respect to the function and activity of the individual subunits.Such information may lead to the identification of compounds which arecapable of very specific interaction with one or more of the receptorsubunits. Such specificity may prove of great value in medicalapplication.

Thus, DNA encoding one or more human neuronal nAChR subunits may beintroduced into suitable host cells (e.g., eukaryotic or prokaryoticcells) for expression of individual subunits and functional nAChRs.Preferably combinations of alpha and beta subunits may be introducedinto cells: such combinations include combinations of any one or more ofα₂, α₃, α₄, α₅, α₆ and α₇ with β₂, β₃ and/or β₄. Sequence informationfor α₅ is presented in Proc. Natl. Acad. Sci. USA (1992) 89:1572–1576;sequence information for α₂, α₃, α₄, α₇, β₂ and β₄ is presented in PCTpublication WO 94/20617, incorporated by reference herein; and sequenceinformation for α₆ and β₃ is presented in the Sequence Listing providedherewith. Presently preferred combinations of subunits include α₆ and/orβ₃ with any one or more of α₂, α₃, α₄, α₅, α₇, β₂ or β₄. It isrecognized that some of the subunits may have ion transport function inthe absence of additional subunits, while others require a combinationof two or more subunits in order to display ion transport function. Forexample, the α₇ subunit is functional in the absence of any added betasubunit. Furthermore, some of the subunits may not form functionalnAChRs alone or in combination, but instead may modulate the propertiesof other nAChR subunit combinations.

In certain embodiments, eukaryotic cells with heterologous humanneuronal nAChRs are produced by introducing into the cell a firstcomposition, which contains at least one RNA transcript that istranslated in the cell into a subunit of a human neuronal nAChR. Inpreferred embodiments, the subunits that are translated include an alphasubunit of a human neuronal nAChR. More preferably, the composition thatis introduced contains an RNA transcript which encodes an alpha subunitand also contains an RNA transcript which encodes a beta subunit of ahuman neuronal nAChR. RNA transcripts can be obtained from cellstransfected with DNAs encoding human neuronal nAChR subunits or by invitro transcription of subunit-encoding DNAs. Methods for in vitrotranscription of cloned DNA and injection of the resulting mRNA intoeukaryotic cells are well known in the art. Amphibian oocytes areparticularly preferred for expression of in vitro transcripts of thehuman neuronal nAChR DNA clones provided herein. See, for example,Dascal (1989) CRC Crit. Rev. Biochem. 22:317–387, for a review of theuse of Xenopus oocytes to study ion channels.

Thus, stepwise introduction into cells of DNA or RNA encoding one ormore alpha subtypes, and one or more beta subtypes is possible. Theresulting cells may be tested by the methods provided herein or known tothose of skill in the art to detect functional nAChR activity. Suchtesting will allow the identification of combinations of alpha and betasubunit subtypes that produce functional nAChRs, as well as individualsubunits that produce functional nAChRs.

As used herein, activity of a human neuronal nAChR refers to anyactivity characteristic of an nAChR. Such activity can typically bemeasured by one or more in vitro methods, and frequently corresponds toan in vivo activity of a human neuronal nAChR. Such activity may bemeasured by any method known to those of skill in the art, such as, forexample, measuring the amount of current which flows through therecombinant channel in response to a stimulus.

Methods to determine the presence and/or activity of human neuronalnAChRs include assays that measure nicotine binding, ⁸⁶Rb ion-flux, Ca²⁺influx, the electrophysiological response of cells, theelectrophysiological response of oocytes injected with RNA, and thelike. In particular, methods are provided herein for the measurement ordetection of an nAChR-mediated response upon contact of cells containingthe DNA or mRNA with a test compound.

As used herein, a recombinant or heterologous human neuronal nAChRrefers to a receptor that contains one or more subunits encoded byheterologous DNA that has been introduced into and expressed in cellscapable of expressing receptor protein. A recombinant human neuronalnAChR may also include subunits that are produced by DNA endogenous tothe host cell. In certain embodiments, recombinant or heterologous humanneuronal nAChR may contain only subunits that are encoded byheterologous DNA.

As used herein, heterologous or foreign DNA and RNA are usedinterchangeably and refer to DNA or RNA that does not occur naturally aspart of the genome of the cell in which it is present or to DNA or RNAwhich is found in a location or locations in the genome that differ fromthat in which it occurs in nature. Typically, heterologous or foreignDNA and RNA refers to DNA or RNA that is not endogenous to the host celland has been artificially introduced into the cell. Examples ofheterologous DNA include DNA that encodes a human neuronal nAChRsubunit, DNA that encodes RNA or proteins that mediate or alterexpression of endogenous DNA by affecting transcription, translation, orother regulatable biochemical processes, and the like. The cell thatexpresses heterologous DNA may contain DNA encoding the same ordifferent expression products. Heterologous DNA need not be expressedand may be integrated into the host cell genome or maintainedepisomally.

Recombinant receptors on recombinant eukaryotic cell surfaces maycontain one or more subunits encoded by the DNA or mRNA encoding humanneuronal nAChR subunits, or may contain a mixture of subunits encoded bythe host cell and subunits encoded by heterologous DNA or mRNA.Recombinant receptors may be homogeneous or may be a mixture ofsubtypes. Mixtures of DNA or mRNA encoding receptors from variousspecies, such as rats and humans, may also be introduced into the cells.Thus, a cell may be prepared that expresses recombinant receptorscontaining only α₆ and β₃ subunits, or in combination with any otheralpha and beta subunits provided herein. For example, either or both ofthe α₆ and β₃ subunits of the present invention can be co-expressed withα₂, α₃, α₄, α₅, α₇, β₂ and/or β₄ receptor subunits. As noted previously,some of the neuronal nAChR subunits may be capable of forming functionalreceptors in the absence of other subunits, thus co-expression is notalways required to produce functional receptors. Moreover, some nAChRsubunits may require co-expression with two or more nAChR subunits toparticipate in functional receptors.

As used herein, a functional neuronal nAChR is a receptor that exhibitsan activity of neuronal nicotinic nAChRs as assessed by any in vitro orin vivo assay disclosed herein or known to those of skill in the art.Possession of any such activity that may be assessed by any method knownto those of skill in the art and provided herein is sufficient todesignate a receptor as functional. Methods for detecting nAChR proteinand/or activity include, for example, assays that measure nicotinebinding, ⁸⁶Rb ion-flux, Ca²⁺ influx, the electrophysiological responseof cells containing heterologous DNA or mRNA encoding one or morereceptor subunit subtypes, and the like. Since all combinations of alphaand beta subunits may not form functional receptors, numerouscombinations of alpha and beta subunits should be tested in order tofully characterize a particular subunit and cells which produce same.Thus, as used herein, “functional” with respect to a recombinant orheterologous human neuronal nAChR means that the receptor channel isable to provide for and regulate entry of human neuronal nAChR-permeableions, such as, for example, Na⁺, K⁺, Ca²⁺ or Ba²⁺, in response to astimulus and/or bind ligands with affinity for the receptor. Preferablysuch human neuronal nAChR activity is distinguishable, such as byelectrophysiological, pharmacological and other means known to those ofskill in the art, from any endogenous nAChR activity that may beproduced by the host cell.

In accordance with a particular embodiment of the present invention,recombinant human neuronal nAChR-expressing mammalian cells or oocytescan be contacted with a test compound, and the modulating effect(s)thereof can then be evaluated by comparing the nAChR-mediated responsein the presence and absence of test compound, or by comparing thenAChR-mediated response of test cells, or control cells (i.e., cellsthat do not express neuronal nAChRs), to the presence of the compound.

As used herein, a compound or signal that “modulates the activity of aneuronal nAChR” refers to a compound or signal that alters the activityof nAChR so that activity of the nAChR is different in the presence ofthe compound or signal than in the absence of the compound or signal. Inparticular, such compounds or signals include agonists and antagonists.The term agonist refers to a substance or signal, such as ACh, thatactivates receptor function; and the term antagonist refers to asubstance that interferes with receptor function. Typically, the effectof an antagonist is observed as a blocking of activation by an agonist.Antagonists include competitive and non-competitive antagonists. Acompetitive antagonist (or competitive blocker) interacts with or nearthe site specific for the agonist (e.g., ligand or neurotransmitter) forthe same or closely situated site. A non-competitive antagonist orblocker inactivates the functioning of the receptor by interacting witha site other than the site that interacts with the agonist.

As understood by those of skill in the art, assay methods foridentifying compounds that modulate human neuronal nAChR activity (e.g.,agonists and antagonists) generally require comparison to a control. Onetype of a “control” cell or “control” culture is a cell or culture thatis treated substantially the same as the cell or culture exposed to thetest compound, except the control culture is not exposed to testcompound. For example, in methods that use voltage clampelectrophysiological procedures, the same cell can be tested in thepresence and absence of test compound, by merely changing the externalsolution bathing the cell. Another type of “control” cell or “control”culture may be a cell or a culture of cells which are identical to thetransfected cells, except the cells employed for the control culture donot express functional human neuronal nAChRs. In this situation, theresponse of test cell to test compound is compared to the response (orlack of response) of receptor-negative (control) cell to test compound,when cells or cultures of each type of cell are exposed to substantiallythe same reaction conditions in the presence of compound being assayed.

Functional recombinant human neuronal nAChRs include at least an alphasubunit, or at least an alpha subunit and a beta subunit of a humanneuronal nAChR. Eukaryotic cells expressing these subunits have beenprepared by injection of RNA transcripts and by transfection of DNA.Such cells have exhibited nAChR activity attributable to human neuronalnAChRs that contain one or more of the heterologous human neuronal nAChRsubunits.

With respect to measurement of the activity of functional heterologoushuman neuronal nAChRs, endogenous nAChR activity and, if desired,activity of nAChRs that contain a mixture of endogenous host cellsubunits and heterologous subunits, should, if possible, be inhibited toa significant extent by chemical, pharmacological andelectrophysiological means.

The invention will now be described in greater detail with reference tothe following non-limiting examples.

EXAMPLE 1 Isolation of DNA Encoding Human nAChR α₆ Subunits

A human substantia nigra cDNA library (Clontech Laboratories, Inc.) wasscreened for hybridization to a fragment of the rat nAChR α₆ subunitcDNA. Isolated plaques were transferred to nitrocellulose filters andhybridization was performed in 5× Denhardt's, 5×SSPE, 50% formamide, 200μg/ml denatured salmon sperm DNA and 0.2% SDS, at 42° C. Washes wereperformed in 0.2×SSPE, 0.2% SDS, at 60° C.

Five hybridizing clones were plaque-purified and characterized byrestriction endonuclease mapping and DNA sequence analysis. The DNAsequence of the 5′- and 3′-ends of the cDNA inserts was determined usingcommercially available λgt10 forward and reverse oligonucleotideprimers. Analysis of the DNA sequence of the five cDNA inserts revealedthat three clones contained the translational initiation codon, afull-length α₆ open reading frame (nucleotides 143–1624 of SEQ ID NO:1),the translational stop codon and 142 additional nucleotides of 5′- and116 nucleotides of 3′-flanking sequences. The amino acid sequencededuced from the nucleotide sequence of the full-length clone has ˜82%identity with the amino acid sequence deduced from the rat nAChR α₆subunit DNA. Several regions of the deduced rat and human α₆ amino acidsequences are notably dissimilar:

-   -   amino acids 1–30 (the human signal sequence has only ˜56%        identity with respect to the rat sequence),

amino acids 31–50 (the human sequence has only ˜70% identity withrespect to the rat sequence),

amino acids 344–391 (the human sequence has only ˜40% identity withrespect to the rat sequence),

amino acids 401–428 (the human sequence has only ˜64% identity withrespect to the rat sequence).

Furthermore, the insert DNA of a single clone, KEα6.5, was determined tobe missing 45 nucleotides of α₆ coding sequence, resulting in anin-frame deletion of 15 amino acid residues of the deduced amino acidsequence (residues 74 to 88 of SEQ ID NO:2). Interestingly, the deducedamino acid sequence immediately downstream of the site of the deletionshares only ˜58% amino acid identity with the deduced rat α₆ amino acidsequence (amino acids 89-100 of SEQ ID NO:2).

EXAMPLE 2 Isolation of DNA Encoding Human Neuronal nAChR β₃ Subunit

A human substantia nigra cDNA library (Clontech Laboratories, Inc.) wasscreened for hybridization to synthetic oligonucleotide primerscomplementary to the C-terminus of human nicotinic nAChR β₃ subunitcDNA. Isolated plaques were transferred to nitrocellulose filters andhybridized under high stringency conditions with respect tooligonucleotides (washing conditions 1×SSPE, 0.2% SDS at 50° C.) withsynthetic oligonucleotide primers complementary to the partial human β₃nAChR subunit described by Willoughby et al., (1993) Neurosci. Lett.155, 136–139.

Two hybridizing clones were plaque-purified and characterized byrestriction endonuclease mapping. The DNA sequence of the 5′- and3′-ends of the cDNA insert was determined using commercially availableT7 and SP6 oligonucleotide primers. The complete sequence of cloneKBβ3.2 was determined. Clone KBβ3.2 contains a 1927 bp cDNA insert thatcontains a 1,377 nucleotide open reading frame encoding a full-length β₃nAChR subunit (nucleotides 98–1472 SEQ ID NO:3) as well as 97nucleotides of 5′- and 453 nucleotides of ₃′-untranslated sequences. Theamino acid sequence deduced from the nucleotide sequence of thefull-length clone has ˜81% identity with the amino acid sequence deducedfrom the rat nicotinic nAChR β₃ subunit DNA. Several regions of thededuced rat and human β₃ amino acid sequences are notably dissimilar:

-   -   amino acids 1–28 (the human signal sequence has only ˜25%        identity with respect to the rat sequence),    -   amino acids 347–393 (the human sequence has only ˜55% identity        with respect to the rat sequence),    -   amino acids 440–464 (the human sequence has only ˜68% identity        with respect to the rat sequence).

EXAMPLE 3 Preparation of Constructs for the Expression of RecombinantHuman Neuronal nAChR Subunits

Isolated cDNAs encoding human neuronal nAChR subunits were incorporatedinto vectors for use in expressing the subunits in mammalian host cellsand for use in generating in vitro transcripts from the DNAs to beexpressed in Xenopus oöcytes. The following vectors were utilized inpreparing the constructs.

A. Constructs for Expressing Human nAChR α₆ Subunits

A 1,743 bp EcoRI fragment, encoding a full-length ACh α₆ subunit, wasisolated from KEα6.3 by standard methods and ligated into the EcoRIpolylinker site of the vector pcDNA3 to generate pcDNA3-KEα6.3 (see FIG.1). Plasmid pcDNA3 (see FIG. 1) is a pUC19-based vector that contains aCMV promoter/enhancer, a T7 bacteriophage RNA polymerase promoterpositioned downstream of the CMV promoter/enhancer, a bovine growthhormone (BGH) polyadenylation signal downstream of the T7 promoter, anda polylinker between the T7 promoter and the BGH polyadenylation signal.This vector thus contains all of the regulatory elements required forexpression in a mammalian host cell of heterologous DNA which has beenincorporated into the vector at the polylinker. In addition, because theT7 promoter is located just upstream of the polylinker, this plasmid canbe used for the synthesis of in vitro transcripts of heterologous DNAthat has been subcloned into the vector at the polylinker. Furthermore,this plasmid contains a gene encoding neomycin resistance used as aselectable marker during transfection. FIG. 1 also shows a partialrestriction map of pcDNA3-KEα6.3.

The expression of the full-length human nAChR α₆ subunit was optimizedby the introduction of a consensus ribosome binding site (RBS; Kozak,1991) prior to the translational start codon. The existing5′-untranslated region was modified by PCR mutagenesis using the plasmidpcDNA3-KEα6.3 as a DNA template and a complementary upstreamoligonucleotide containing the appropriate nucleotide RBS substitutionsas well as flanking 5′ HindIII and EcoRI sites, and an oligonucleotidecomplementary to α₆ coding sequences ˜450 nucleotides downstream of thetranslational start codon. The resulting PCR fragment contained HindIIIand EcoRI sites followed by the consensus RBS and nucleotides 1–459 ofthe human ACh α₆ coding sequence (nucleotides 143–602 of SEQ ID NO:1).The amplified DNA was digested with HindIII and BamHI; the 308 bpHindIII-BamHI fragment was isolated and ligated with the 5.3 kbBamHI-PvuI fragment of pcDNA3-KE6.3 and the 1.4 kb PvuI to HindIIIfragment from pcDNA3 to generate the ˜7.0 kb plasmid pcDNA3-KEα6RBS.

B. Constructs for Expressing Human Neuronal nAChR β₃ Subunits

An ˜2.0 kb EcoRI fragment, encoding a full-length nicotinic ACh β₃subunit, was isolated from KEβ3.2 by standard methods and ligated intothe EcoRI polylinker site of the vector pcDNA3 to generate pcDNA3-KEβ3.2(see FIG. 2). FIG. 2 also shows a partial restriction map ofpcDNA3-KEβ3.2.

The expression of the full-length human nicotinic nAChR β₃ subunit isoptimized by the introduction of a consensus ribosome binding site (RBS)prior to the translational start codon. The existing 5′-untranslatedregion is modified by PCR mutagenesis using a method similar to thatdescribed above for the α₆ nAChR subunit.

EXAMPLE 4 Expression of Recombinant Human Neuronal nAChR in Oocytes

Xenopus oocytes are injected with in vitro transcripts prepared fromconstructs containing DNA encoding α₆ and β₃ subunits.Electrophysiological measurements of the oocyte transmembrane currentsare made using the two-electrode voltage clamp technique (see, e.g.,Stuhmer (1992) Meth. Enzymol. 207:319–339).

1. Preparation of in vitro Transcripts

Recombinant capped transcripts of pcDNA3-KEα6RBS and pcDNA3-KBβ3RBS aresynthesized from linearized plasmids using the mMessage and mMachine invitro transcription kit according to the capped transcript protocolprovided by the manufacturer (Catalog 1344 from AMBION, Inc., Austin,Tex.). The mass of each synthesized transcript is determined by UVabsorbance and the integrity of each transcript is determined byelectrophoresis through an agarose gel.

2. Electrophysiology

Xenopus oocytes are injected with either 12.5, 50 or 125 ng of one ormore human nicotinic nAChR α and β subunit transcript per oocyte. Thepreparation and injection of oocytes is carried out as described byDascal (1987) in Crit. Rev. Biochem. 22:317–387. Two-to-six daysfollowing mRNA injection, the oocytes are examined using thetwo-electrode voltage clamp technique. The cells are bathed in Ringer'ssolution (115 mM NaCl, 2.5 mM KCl, 1.8 mM CaCl₂, 10 mM HEPES, pH 7.3)containing 1 μM atropine with or without 100 μM d-tubocurarine. Cellsare seen to be voltage-clamped at −60 to −80 mV. Data are acquired withAxotape software at 2–5 Hz. The agonists acetylcholine (ACh), nicotine,and cytisine are added at concentrations ranging from 0.1 μM to 100 μM.

EXAMPLE 5 Recombinant Expression of Human nAChR Subunits in MammalianCells

Human embryonic kidney (HEK) 293 cells are transiently and stablytransfected with DNA encoding human neuronal nicotinic nAChR α₆ and β₃subunits. Transient transfectants are analyzed for expression ofnicotinic nAChR using various assays, e.g., electrophysiologicalmethods, Ca²⁺-sensitive fluorescent indicator-based assays.

1. Transient Transfection of HEK Cells

HEK cells are transiently co-transfected with DNA encoding one or more asubunit and/or one or more subunits. Approximately 2×10⁶ HEK cells aretransiently transfected with 18 μg of the indicated plasmid(s) accordingto standard CaPO₄ transfection procedures [Wigler et al. (1979) Proc.Natl. Acad. Sci. USA 76:1373–1376] or using lipofectamine according tothe manufacturer's instructions (Bethesda Research Laboratory (BRL),Gaithersburg, Md.). In addition, 2 μg of plasmid pCMVβgal (ClontechLaboratories, Palo Alto, Calif.), which contains the Escherichia coliβ-galactosidase gene fused to the CMV promoter, are co-transfected as areporter gene for monitoring the efficiency of transfection. Thetransfectants are analyzed for β-galactosidase expression by measurementof β-galactosidase activity [Miller (1972) Experiments in MolecularGenetics, pp.352–355, Cold Spring Harbor Press]. Transfectants can alsobe analyzed for β-galactosidase expression by direct staining of theproduct of a reaction involving β-galactosidase and the X-gal substrate[Jones (1986) EMBO 5:3133–3142].

2. Stable Transfection of HEK Cells

HEK cells are transfected using the calcium phosphate transfectionprocedure [Current Protocols in Molecular Biology, Vol. 1, WileyInter-Science, Supplement 14, Unit 9.1.1–9.1.9 (1990)]. HEK cells aretransfected with 1 ml of DNA/calcium phosphate precipitate containingthe DNA encoding the desired alpha and beta subunits and pSV2neo (as aselectable marker). After 14 days of growth in medium containing 1 μg/mlG418, colonies form and are individually isolated by using cloningcylinders. The isolates are subjected to limiting dilution and screenedto identify those that expressed the highest level of nAChR, asdescribed below.

3. Analysis of Transfectants

a. Fluorescent Indicator-based Assays

Activation of the ligand-gated nAChR by agonists leads to an influx ofcations, including Ca⁺⁺, through the receptor channel. Ca⁺⁺ entry intothe cell through the channel can induce release of calcium contained inintracellular stores. Monovalent cation entry into the cell through thechannel can also result in an increase in cytoplasmic Ca⁺⁺ levelsthrough depolarization of the membrane and subsequent activation ofvoltage-dependent calcium channels. Therefore, methods of detectingtransient increases in intracellular calcium concentration can beapplied to the analysis of functional nicotinic nAChR expression. Onemethod for measuring intracellular calcium levels relies oncalcium-sensitive fluorescent indicators.

Calcium-sensitive indicators, such as fluo-3 (Catalog No. F-1241,Molecular Probes, Inc., Eugene, Oreg.), are available as acetoxymethylesters which are membrane permeable. When the acetoxymethyl ester formof the indicator enters a cell, the ester group is removed by cytosolicesterases, thereby trapping the free indicator in the cytosol.Interaction of the free indicator with calcium results in increasedfluorescence of the indicator; therefore, an increase in theintracellular Ca²⁺ concentration of cells containing the indicator canbe expressed directly as an increase in fluorescence. An automatedfluorescence detection system for assaying nicotinic nAChR has beendescribed in commonly assigned pending U.S. patent application Ser. No.07/812,254 and corresponding PCT Patent Application No. US92/11090.

HEK cells that are transiently or stably co-transfected with DNAencoding appropriate α and/or β subunits and α₆ and β₃ subunits areanalyzed for expression of functional recombinant nAChR using theautomated fluorescent indicator-based assay. The assay procedure is asfollows.

Untransfected HEK cells and HEK cells co-transfected with theappropriate α and β subunits are plated in the wells of a 96-wellmicrotiter dish and loaded with fluo-3 by incubation for 2 hours at 20°C. in a medium containing 20 μM fluo-3, 0.2% Pluronic F-127 in HBS (125mM NaCl, 5 mM KCl, 1.8 mM CaCl₂, 0.62, mM MgSO₄, 6 mM glucose, 20 mMHEPES, pH 7.4). The cells are then washed with assay buffer (i.e., HBS).The antagonist d-tubocurarine is added to some of the wells at a finalconcentration of 10 μM. The microtiter dish is then placed into afluorescence plate reader and the basal fluorescence of each well ismeasured and recorded before addition of agonist, e.g., 200 μM nicotine,to the wells. The fluorescence of the wells is monitored repeatedlyduring a period of approximately 60 seconds following addition ofnicotine.

The fluorescence of the untransfected HEK cells does not change afteraddition of nicotine. In contrast, the fluorescence of theco-transfected cells, in the absence of d-tubocurarine, increasesdramatically after addition of nicotine to the wells. Thisnicotine-stimulated increase in fluorescence was not observed inco-transfected cells that had been exposed to the antagonistd-tubocurarine. These results demonstrate that the co-transfected cellsexpress functional recombinant nAChR that are activated by nicotine andblocked by d-tubocurarine.

EXAMPLE 6 Characterization of Cell Lines Expressing Human NeuronalnAChRs

Recombinant cell lines generated by transfection with DNA encoding humanneuronal nAChRs, such as those described in Example 3, can be furthercharacterized using one or more of the following methods.

A. Northern or Slot Blot Analysis for Expression of α- and/or β-SubunitEncoding Messages

Total RNA is isolated from ˜1×10⁷ cells and 10–15 μg of RNA from eachcell type is used for Northern or slot blot hybridization analysis. Theinserts from human neuronal nAChR-encoding plasmids can benick-translated and used as probe. In addition, a fragment of theglyceraldehyde-3-phosphate dehydrogenase (GAPD) gene sequence (Tso etal. (1985) Nucleic Acids Res. 13, 2485) can be nick-translated and usedas a control probe on duplicate filters to confirm the presence orabsence of RNA on each blot and to provide a rough standard for use inquantitating differences in α- or β-specific mRNA levels between celllines. Typical Northern and slot blot hybridization and wash conditionsare as follows:

-   -   hybridization in 5×SSPE, 5× Denhardt's solution, 0.2% SDS, 200        μg/ml denatured, sonicated herring sperm DNA, 50% formamide, at        42° C. followed by washing in 0.1×SSPE, 0.1% SDS, at 65° C.

B. Nicotine-binding Assay

Cell lines generated by transfection with human neuronal nAChR α- or α-and β-subunit-encoding DNA can be analyzed for their ability to bindnicotine, for example, as compared to control cell lines:

-   -   neuronally-derived cell lines PC12 (Boulter et al., (1986),        supra; ATCC #CRL1721) and IMR32 (Clementi, et al. (1986);        Int. J. Neurochem. 47:291–297; ATCC #CCL127), and muscle-derived        cell line BC3H1 (Patrick, et al., (1977); J. Biol. Chem.        252:2143–2153). Negative control cells (i.e., host cells from        which the transfectants were prepared) are also included in the        assay. The assay is conducted as follows:

Just prior to being assayed, transfected cells are removed from platesby scraping. Positive control cells used are PC12, BC3H1, and IMR32(which had been starved for fresh media for seven days). Control celllines are removed by rinsing in 37° C. assay buffer (50 mM Tris/HCl, 1mM MgCl₂, 2 mM CaCl₂, 120 mM NaCl, 3 mM EDTA, 2 mg/ml BSA and 0.1%aprotinin at pH7.4). The cells are washed and resuspended to aconcentration of 1×10⁶/250 μl. To each plastic assay tube is added 250μl of the cell solution, 15 nM ³H-nicotine, with or without 1 mMunlabeled nicotine, and assay buffer to make a final volume of 500 μl.The assays for the transfected cell lines are incubated for 30 min atroom temperature; the assays of the positive control cells are incubatedfor 2 min at 1° C. After the appropriate incubation time, 450 μlaliquots of assay volume are filtered through Whatman GF/C glass fiberfilters which has been pretreated by incubation in 0.05%polyethyleneimine for 24 hours at 4° C. The filters are then washedtwice, with 4 ml each wash, with ice cold assay buffer. After washing,the filters are dried, added to vials containing 5 ml scintillationfluid and radioactivity is measured.

C. ⁸⁶Rb Ion-flux Assay

The ability of nicotine or nAChR agonists and antagonists to mediate theinflux of ⁸⁶Rb into transfected and control cells has been found toprovide an indication of the presence of functional nAChRs on the cellsurface. The ⁸⁶Rb ion-flux assay is conducted as follows:

1. The night before the experiment, cells are plated at 2×10⁶ per well(i.e., 2 ml per well) in a 6-well polylysine-coated plate.

2. The culture medium is decanted and the plate washed with 2 ml ofassay buffer (50 mM HEPES, 260 mM sucrose, 5.4 mM KCl, 1.8 mM CaCl₂, 0.8mM MgSO₄, 5.5. mM glucose) at room temperature.

3. The assay buffer is, decanted and 1 ml of assay buffer, containing 3μCi/ml ⁸⁶Rb, with 5 mM ouabain and agonist or antagonist in aconcentration to effect a maximum response, is added.

4. The plate is incubated on ice at 1° C. for 4 min.

5. The buffer is decanted into a waste container and each well waswashed with 3 ml of assay buffer, followed by two washes of 2 ml each.

6. The cells are lysed with 2×0.5 ml of 0.2% SDS per well andtransferred to a scintillation vial containing 5 ml of scintillationfluid.

7. The radioactivity contained in each vial is measured and the datacalculated.

Positive control cells provided the following data in this assay:

PC12 IMR32 Maximum Maximum EC₅₀ response EC₅₀ response Agonist nicotine52 μM 2.1X^(a) 18 μM 7.7X^(a) CCh* 35 μM 3.3X^(b) 230 μM 7.6X^(c)cytisine 57 μM 3.6X^(d) 14 μM 10X^(e) Antagonist d-tubocurarine 0.81 μM2.5 μM mecamylamine 0.42 μM 0.11 μM hexamethonium nd^(f) 22 μM atropine12.5 μM 43 μM *CCh = carbamylcholine ^(a)200 μM nicotine ^(b)300 μM CCh^(c)3 mM CCh ^(d)1 mM cytisine ^(e)100 μM cytisine ^(f)nd = notdetermined

D. Electrophysiological Analysis of Mammalian Cells Transfected withHuman Neuronal nAChR Subunit-encoding DNA

Electrophysiological measurements may be used to assess the activity ofrecombinant receptors or to assess the ability of a test compound topotentiate, antagonize or otherwise modulate the magnitude and durationof the flow of cations through the ligand-gated recombinant nAChR. Thefunction of the expressed neuronal nAChR can be assessed by a variety ofelectrophysiological techniques, including two-electrode voltage clampand patch clamp methods. The cation-conducting channel intrinsic to thenAChR opens in response to acetylcholine (ACh) or other nicotiniccholinergic agonists, permitting the flow of transmembrane currentcarried predominantly by sodium and potassium ions under physiologicalconditions. This current can be monitored directly by voltage clamptechniques. In preferred embodiments, transfected mammalian cells orinjected oocytes are analyzed electrophysiologically for the presence ofnAChR agonist-dependent currents.

While the invention has been described in detail with reference tocertain preferred embodiments thereof, it will be understood thatmodifications and variations are within the spirit and scope of thatwhich is described and claimed.

Summary of Sequences

Sequence ID No. 1 is a nucleotide sequence encoding an α₆ subunit ofhuman neuronal nicotinic acetylcholine receptor, and the deduced aminoacid sequence thereof.

Sequence ID No. 2 is the amino acid sequence of the α₆ subunit of humanneuronal nicotinic acetylcholine receptor set forth in Sequence ID No.1.

Sequence ID No. 3 is a nucleotide sequence encoding a β₃ subunit ofhuman neuronal nicotinic acetylcholine receptor, and the deduced aminoacid sequence thereof.

Sequence ID No. 4 is the amino acid sequence of the β₃ subunit of humanneuronal nicotinic acetylcholine receptor set forth in Sequence ID No.3.

1. A substantially pure human neuronal nicotinic acetylcholine α₆subunit encoded by a nucleic acid molecule comprising nucleotides143–1624 of SEQ ID NO:1.
 2. A substantially pure human neuronalnicotinic acetylcholine α₆ subunit encoded by a nucleic acid molecule asset forth in SEQ ID NO:1.
 3. A substantially pure human neuronalnicotinic acetylcholine receptor, comprising an α₆ human neuronalnicotinic acetylcholine receptor subunit, wherein the subunit comprisesthe amino acid sequence as set forth in SEQ ID NO:2.
 4. The nicotinicreceptor of claim 3, further comprising a human neuronal nicotinicacetylcholine receptor β subunit.
 5. A substantially pure human neuronalnicotinic acetylcholine receptor β₃ subunit, wherein the subunit isencoded by a nucleic acid molecule comprising nucleotides 98–1471 as setforth in SEQ ID NO:3.
 6. A substantially pure human neuronal nicotinicacetylcholine β₃ subunit comprising the sequence of amino acids as setforth in SEQ ID NO:4.
 7. A substantially pure recombinant human neuronalnicotinic acetylcholine receptor, comprising a β₃ human neuronalnicotinic acetylcholine receptor subunit encoded by the nucleic acidmolecule of claim
 5. 8. The neuronal nicotinic acetylcholine receptor ofclaim 6, further comprising at least one human neuronal nicotinicacetylcholine receptor α subunit.